ATP-sensitive potassium channels (K+ channels), which represent a family of potassium channels inhibited by intracellular ATP, have been found in a variety of tissues including heart, pancreatic beta-cells, skeletal muscle, smooth muscle and the central nervous system (G. Edwards and A. H. Weston, Ann. Rev. Pharmacol. Toxicol. 33: 597-637 (1993)). These ATP-sensitive K+ channels have been associated with diverse cellular functions, such as shortening of action potential duration and cellular loss of potassium ions that occur during metabolic inhibition in heart, smooth muscle relaxation, regulation of skeletal muscle excitability, and neurotransmitter release (A. Terzic, et al., Am. J. Physiol. 269: C525-C545 (1995)).
For example, in pancreatic beta-cells (β-cells), ATP-sensitive K+ channels play an important role in linking the metabolic status of the cell to cellular excitability. The primary physiological stimulus for insulin secretion is a rise in blood glucose concentrations. Glucose enters the beta-cell where it is metabolized resulting in elevated intracellular ATP and a concomitant lowering of intracellular ADP. These changes in nucleotide levels act synergistically to close ATP-sensitive K+ channels in the plasma membrane, because ATP inhibits whereas ADP activates channel activity. The closure of ATP-sensitive K+ channels causes a membrane depolarization that opens voltage dependent calcium channels and triggers electrical activity. The calcium influx that ensues raises intracellular calcium and stimulates insulin secretion (F. M. Ashcroft, Nature Medicine, 2: 1301-1302 (1996)). Recently, it has been shown that the ATP-sensitive K+ channel in pancreas is a complex composed of at least two subunits, a channel forming subunit (Kir 6.2) that selectively conducts potassium and a regulatory protein termed the sulfonylurea receptor (SUR1) (N. Inagaki, et al., Science 270: 1166-1170). Co-expression of these two subunits constituted inwardly rectifying ATP-sensitive K+ channels with expected pharmacological and biophysical properties.
An emerging diversity of ATP-sensitive K+ channels is now known to exist. Two channel forming subunits (Kir 6.1 and Kir 6.2) and three regulatory subunits (SUR 1, SUR 2A and SUR 2B) have recently been cloned from mammalian tissues (S. Isomoto, et al., Neuron 16: 1011-1017 (1996)). The elucidation of this molecular diversity supports earlier pharmacological studies demonstrating that ATP-sensitive K+ channels in different tissues exhibit considerable variation in response to activators and inhibitors (G. Edwards, supra).
ATP-sensitive potassium channels are the molecular targets for two important classes of drugs, the sulfonylureas and the K+ channel openers. Sulfonylureas are widely used in the management of non-insulin dependent diabetes mellitus (NIDDM), a disease characterized by decreased insulin content and impaired response to glucose (H. E. Lebovitz, in Diabetes Mellitus: Theory and Practice, eds. H. Rifkin, & D. Porte, Jr. (Elsevier, N.Y.), pp.554-574 (1990)). In the pancreas, sulfonylurea drugs stimulate insulin secretion in the islets of Langerhans, which have partially lost their sensitivity to glucose (E. Cerasi, et al., Diabetes 21: 224-234 (1972)). This class of drugs inhibits ATP-sensitive K+ channel opening through an interaction with the regulatory subunit (SUR 1) of the pancreatic β-cell ATP-sensitive K+ channel and thereby stimulate insulin release (E. Cerasi, et al., Diabetes 21: 224-234 (1972)).
The biological effects of sulfonylurea drugs led Virsolvy-Vergine, et al. to postulate the existence of an endogenous ligand for sulfonylurea receptors (A. Virsolvy-Vergine, et al., FEBS Lett. 242: 65-69 (1988)). Later they identified a peptide from ovine brain, which was shown to bind receptors from both the central nervous system (CNS) and pancreatic β cells and to induce insulin secretion from a rodent beta-cell tumor line (βTC cells) in vitro. They concluded that this peptide is a natural ligand for the sulfonylurea receptor and may play a role in the normal physiology of the CNS and pancreas. They termed this peptide “endosulfine” (A. Virsolvy-Vergine, et al., Proc. Natl. Acad. Sci. USA 89: 6629-6633 (1992)).
The complete amino acid or nucleotide sequence of endosulfine has not been determined in any species; partial cDNA sequence has recently been obtained from bovine tissue (K. Peyrollier, et al., Biochem. Biophys. Res. Comm. 223: 583-586 (1996)). No information on the nucleotide or amino acid sequence of human endosulfine has been reported. The emerging molecular diversity of ATP-senstive K+ channels raises the possibility that a family of endosulfine molecules may exist, that display tissue specific expression and differentially interact with SUR isoforms.
The reported effects of bovine and ovine endosulfine to interact with sulfonylurea receptors and modulate insulin release suggests that the isolation and characterization of a family of human endosulfines offers the potential to develop new therapeutic and diagnostic agents for the treatment of disease states. Such therapeutics may include recombinant endosulfine, antisense deoxyribonucleotides, transcriptional regulators and activators or inhibitors of endosulfine activity. For example, human endosulfine or modulators of endosulfine activity may represent an alternative approach to the treatment of diabetes if a human endosulfine is found to modulate insulin levels as has been reported for ovine endosulfine (A. Virsolvy-Vergine, et al., (1992) supra). Furthermore, in the CNS, where ATP-sensitive K+ channels modulate neuronal excitability, modulators of endosulfine may represent a target for the treatment of disease involving abnormal neuronal firing such as epilepsy, pain, depression and ischemia. In heart, where SUR 2A is selectively expressed and ATP-sensitive K+ channels play a role in control of action potential duration, modulation of endosulfine levels and/or activity may represent an approach to the treatment of cardiac ischemia. ATP-sensitive K+ channels expressed in skeletal muscle alter the electrical activity of the cells in response to changes in energy status and modulation of this complex with K+ channel openers has proven beneficial in a number of disease states including myotonia congita and for patients with hypokalemic paralysis (A. C. Wareham, in Potassium Channel Modulators: Pharmacological, Molecular and Clinical Aspects: eds. A. H. Weston, and T. C. Hamilton, pp. 110-143 (1992)). However in this case, the side effects of the compounds precludes their widespread use. An endosulfine selectively expressed in skeletal muscle may offer an alternative approach to normalizing skeletal muscle excitability and thus may be utilized in a number of skeletal muscle disorders. Therefore, it would be advantageous to isolate of a DNA sequence encoding a full length human endosulfine which would permit investigation of its use toward the development of therapeutics.
Mutations in SUR1 and Kir 6.2 have been linked to persistant hyperinsulinemic hypoglycernia of infancy (PHHI) (P. M. Thomas, et al. Science 268: 426-429 (1995) and P. Thomas, et al., Hum. Mol. Gen. 5: 1809-1812 (1996)) and a recent report has suggested that mutations in SUR1 may also be linked to non-insulin dependent diabetes (H. Inoue, et al., Diabetes 45: 825-831, (1996)). The isolation and characterization of a human endosulfine will facilitate studies to determine if mutations exist in the endosulfine gene, if such mutations result in altered expression and/or activity of the protein and if these mutations are linked to disease states such as diabetes, epilepsy, depression and ischemia. The ability to identify normal and mutated forms of endosulfine in human cells would then offer the opportunity to develop diagnostic tests for such disease states.
Thus, it would be advantageous to provide specific methods and reagents for the diagnosis, staging, prognosis, monitoring, prevention or treatment of diseases and conditions associated with abnormal regulation or expression of human endosulfine or to indicate possible predisposition to these conditions.